Abstract 6313: IND-enabling studies for GrB-Fc-IT4: <i>in vivo</i> efficacy, pharmacokinetics, schedule optimization and maximum tolerated dose
Authors: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum
Journal: Cancer Research (2023)

Abstract Fn14 (fibroblast growth factor-inducible 14) is the only known receptor of TWEAK (tumor necrosis factor-related weak inducer of apoptosis). The binding of TWEAK to Fn14 activates several intracellular signal transduction cascades that lead to cell death, proliferation, migration, or survival depending on the cellular contexts. Several studies have shown that Fn14 is upregulated in many solid tumors when compared with healthy tissues. The activation of TWEAK/Fn14 signaling increases the proliferation, invasion, and migration of tumor cells. Moreover, angiogenesis, pro-inflammatory cytokine expression, and epithelial-mesenchymal transitions are promoted upon TWEAK/Fn14 activation. Therefore, Fn14 is considered a potential therapeutic target, and attempts to generate targeted therapeutics have achieved limited success in pre-clinical and clinical trials. We generated a completely human fusion protein, GrB-Fc-IT4, consisting of the IT4 scFv antibody targeting the Fn14 receptor and the serine protease granzyme B (GrB) as the cytotoxic payload. Comprehensive mechanism of action studies showed that GrB-Fc-IT4 effectively induced cell death in vitro against a broad selection of tumor types with high specificity. The intracellular pathway included rapid, irreversible activation of the caspase cascade and independent mitochondrial depolarization leading to intense apoptotic cellular damage. Pharmacokinetic studies in mice showed that GrB-Fc-IT4 cleared bi-exponentially from plasma with a rapid initial clearance (t ½α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2β = 35 hours), similar to that of IgGs. Based on our PK study, we compared the in vivo antitumor efficacy of GrB-Fc-IT4 against A549 (NSCLC) tumors, using two different dosing schedules (QODx5 vs QWx5). Tumor efficacy showed a clear dose and schedule dependence. Mice bearing A549 tumors treated with 32 mg/kg/dose (QOWx5) exhibited complete tumor growth inhibition (7/10 tumors regressed) with 3/10 tumors showing no growth up to 50 days after the last dose. Thus, GrB-Fc-IT4 displays impressive in vitro and in vivo cytotoxic effects. Even though GrB-Fc-IT4 cross-reacts with the murine Fn14 antigen, maximum tolerated dose studies in mice have confirmed that GrB-Fc-IT4 is a safe product, as mice treated with the drug up to 500 mg/kg showed no evidence of toxicity (changes in respiration, rough hair coat, unusual behavior or hunched posture). Taking in consideration these results we estimate that the therapeutic index for this class of drug is higher than four. Our data suggest that GrB-Fc-IT4 is a novel antitumor agent with a unique mechanism of action compared to all other therapeutic agents. The exceptionally safe and effective profile of this drug makes it an ideal candidate for advancing to clinical trials. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Mihai Gagea, Michael G. Rosenblum. IND-enabling studies for GrB-Fc-IT4: in vivo efficacy, pharmacokinetics, schedule optimization and maximum tolerated dose [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6313.